Project Summary/Abstract The goal of this proposal is to investigate cocaine-mediated regulation of astrocyte mitochondrial antiviral signaling (MAVS) protein via mitochondrial toxicity; thereby resulting in differential modulation of viral-mediated neuroinflammation and innate immune responses.Cocaine is the second highest used illegal drug in the US, decreasing the users ability to fight off infections and increasing the severity and onset of viral-mediated neuroinvasion and neurotoxicity [1, 2]. Astrocytes are the first line of defense against neurotoxicity associated with cocaine abuse and invading pathogens, becoming activated and initiating a robust innate immune response to pathogens [3-6]. Astrocyte release of proinflammatory cytokines, upon cocaine treatment is well documented [12, 13], however; the molecular mechanisms and regulation of cocaine on antiviral responses, thereby impacting viral-induced innate immunity, remains elusive. Our preliminary data, in human astrocytes treated with polyinosinic:polycytidylic acid, (poly I:C, a synthetic dsRNA virus) and infected with Zika virus (ZIKV, neurotrophic ssRNA virus), demonstrates that cocaine alone does not increase astrocyte inflammatory responses, but robustly increases IFN? levels alone, suggesting activation of interferon response factor (IRF) signaling [11]. Cocaine exacerbates poly I:C-induced interferon stimulated response element (ISRE) promoter activity and upregulates ZIKV and poly I:C-mediated innate immune responses. Interestingly, we observed cocaine-mediated regulation of astrocyte mitochondrial fission/fusion proteins, membrane permeability and neuroinflammatory changes with antioxidants; suggesting generation of oxidative stress; which are outcomes identified to regulate mitochondrial antiviral signaling (MAVS) protein function, a mitochondrial scaffolding protein that initiates IRF and NF-?B signaling via recruitment of TNF associated factor (TRAF) proteins [28[14] [15] [16]. Lastly, cocaine reduces cleavage of astrocyte MAVS and increases aggregation of MAVS in SVGA astrocytes and normal human astrocytes (NHA), which is reported to result in a preferential activation of IRF signaling and decreased NF-?B signaling. We hypothesize that cocaine is a critical regulator of astrocyte MAVS; therefore, impacting viral-induced innate immune responses. Furthermore, cocaine-mediated regulation of mitochondrial dynamics modulates MAVS expression, localization and differentially modulates activation of downstream IRF3/7 and NF-?B activation and translocation, ultimately resulting in attenuated responses to CNS viral infection. We will conduct investigations in the molecular regulation of MAVS following cocaine-mediated mitochondrial dysregulation in Aim 1 and delineate the effects of cocaine- mediated astrocyte MAVS regulation during comorbidity in viral CNS infection in Aim 2. Astrocyte MAVS regulation by cocaine is highly significant, in that it may dictate the balance of viral- induced activation of astrocyte innate immune responses having larger implications in innate immune responses to CNS viral infections during cocaine use. !